Eric Quemeneur

Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition

ABSTRACT We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro. Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8+ and CD4+). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs.

Immune checkpoint blockade, immunogenic chemotherapy or IFN-α blockade boost the local and abscopal effects of oncolytic virotherapy

Athough the clinical efficacy of oncolytic viruses has been demonstrated for local treatment, the ability to induce immune-mediated regression of distant metastases is still poorly documented. We report here that the engineered oncolytic vaccinia virus VVWR-TK-RR--Fcu1 can induce immunogenic cell death and generate a systemic immune response. Effects on tumor growth and survival was largely driven by CD8+ T cells, and immune cell infiltrate in the tumor could be reprogrammed toward a higher ratio of effector T cells to regulatory CD4+ T cells. The key role of type 1 IFN pathway in oncolytic virotherapy was also highlighted, as we observed a strong abscopal response in Ifnar-/- tumors. In this model, single administration of virus directly into the tumors on one flank led to regression in the contralateral flank. Moreover, these effects were further enhanced when oncolytic treatment was combined with immunogenic chemotherapy or with immune checkpoint blockade. Taken together, our results suggest how to safely improve the efficacy of local oncolytic virotherapy in patients whose tumors are characterized by dysregulated IFNα signaling. Cancer Res; 77(15); 4146-57. ©2017 AACR.

Cowpox Virus: A New and Armed Oncolytic Poxvirus

Oncolytic virus therapy has recently been recognized as a promising new therapeutic approach for cancer treatment. In this study, we are proposing for the first time to evaluate the in vitro and in vivo oncolytic capacities of the Cowpox virus (CPXV). To improve the tumor selectivity and oncolytic activity, we developed a thymidine kinase (TK)-deleted CPXV expressing the suicide gene FCU1, which converts the non-toxic prodrug 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) and 5-fluorouridine-5′-monophosphate (5-FUMP). This TK-deleted virus replicated efficiently in human tumor cell lines; however, it was notably attenuated in normal primary cells, thus displaying a good therapeutic index. Furthermore, this new recombinant poxvirus rendered cells sensitive to 5-FC. In vivo, after systemic injection in mice, the TK-deleted variant caused significantly less mortality than the wild-type strain. A biodistribution study demonstrated high tumor selectivity and low accumulation in normal tissues. In human xenograft models of solid tumors, the recombinant CPXV also displayed high replication, inducing relevant tumor growth inhibition. This anti-tumor effect was improved by 5-FC co-administration. These results demonstrated that CPXV is a promising oncolytic vector capable of expressing functional therapeutic transgenes.

Viral based vaccine TG4010 induces broadening of specific immune response and improves outcome in advanced NSCLC

BackgroundAdvanced non-small cell lung cancer patients receiving TG4010, a therapeutic viral vaccine encoding human Mucin 1 and interleukin-2 in addition to standard chemotherapy, displayed longer overall survival in comparison to that of patients treated with standard chemotherapy alone. Our study intended to establish the association between overall survival and vaccine-induced T cell responses against tumor associated antigens (TAA) targeted by the vaccine.MethodThe TIME trial was a placebo-controlled, randomized phase II study aimed at assessing efficacy of TG4010 with chemotherapy in NSCLC. 78 patients from the TIME study carrying the HLA-A02*01 haplotype were analyzed using combinatorial encoding of MHC multimers to detect low frequencies of cellular immune responses to TG4010 and other unrelated TAA.ResultsWe report that improvement of survival under TG4010 treatment correlated with development of T cell responses against MUC1. Interestingly, responses against MUC1 were associated with broadening of CD8 responses against non-targeted TAA, thus demonstrating induction of epitope spreading.ConclusionOur results support the causality of specific T-cell response in improved survival in NSCLC. Additionally, vaccine induced epitope spreading to other TAA participates to the enrichment of the diversity of the anti-tumor response. Hence, TG4010 appears as a useful therapeutic option to maximize response rate and clinical benefit in association with other targeted immuno-modulators.Trial registrationRegistered on ClinicalTrials.gov under identifier NCT01383148 on June 23rd, 2011.

Sequential administration of MVA-based vaccines and PD-1/PD-L1-blocking antibodies confers measurable benefits on tumor growth and survival: Preclinical studies with MVA-βGal and MVA-MUC1 (TG4010) in a murine tumor model

ABSTRACT TG4010, a Modified Vaccinia virus Ankara (MVA) expressing human mucin1 (MUC1) has demonstrated clinical benefit for patients suffering from advanced non-small cell lung cancer (NSCLC) in combination with chemotherapy. To support its development, preclinical experiments were performed with either TG4010 or β-galactosidase-encoding MVA vector (MVA-βgal) in mice presenting tumors in the lung. Tumor growth was obtained after intravenous injection of CT26 murine colon cancer cells, engineered to express either MUC1 or βgal. Mice showed increased survival rates after repeated intravenous injections of TG4010 or MVA-βgal, compared to an empty MVA control vector. Treatment with MVA vectors led to the accumulation of CD3dimCD8dim T cells, with two subpopulations characterized as KLRG1+CD127− short-lived effector cells (SLECs), and KLRG1−CD127− early effector cells (EECs) comprising cells releasing IFNγ, Granzyme B and CD107a upon antigen-specific peptide stimulation. EECs were characterized by an up-regulation of PD-1. Tumor growth in the diseased lung correlated with the appearance of PD1+ Treg cells that partially disappeared after TG4010 treatment. At late stage of tumor development in the lung, PD-L1 was detected on CD45− tumor cells, on CD4+ cells, including Treg cells, on CD3+CD8+ and CD3dimCD8dim T lymphocytes, on NK cells, on MDSCs and on alveolar macrophages. We demonstrated that targeting the PD-1/PD-L1 pathway with blocking monoclonal antibodies several days after TG4010 treatment, at late stage of tumor development, enhanced the therapeutic protection induced by the vaccine, supporting the ongoing clinical evaluation of TG4010 immunotherapy in combination with Nivolumab.

Modified Vaccinia virus Ankara-based vaccines in the era of personalized immunotherapy of cancer

ABSTRACT While interest in immunotherapies is renewed by the successful introduction of immune checkpoint blocking agent in the clinic, advances in genome sequencing are opening new possibilities in the design of increasingly personalized vaccines. Personalization of medicine can now be realistically contemplated at the single patient level. Unlike the previous generation of cancer vaccines, neoantigen directed vaccines would target truly specific tumor antigens resulting from acquired tumor genome mutations. Immune response induced by this next generation vaccine would not be subject to self-tolerance and will likely result to enhanced efficacy. Nevertheless, this new technologies can hold to their promises only if sponsors manage to meet several scientific, technical, logistical and regulatory challenges. In particular manufacturers will have to design, manufacture, and deliver to the patient a new pharmaceutical grade in a matters of weeks. In this paper, we briefly review current technologies currently tried at the translation of personalized vaccines and explore the possibilities offered by the Modified Vaccinia virus Ankara in this next wave of cancer vaccines.

Vaccinia Virus Shuffling: deVV5, a Novel Chimeric Poxvirus with Improved Oncolytic Potency

Oncolytic virus (OV) therapy has emerged as a promising approach for cancer treatment with the potential to be less toxic and more efficient than classic cancer therapies. Various types of OVs in clinical development, including Vaccinia virus (VACV)-derived OVs, have shown good safety profiles, but limited therapeutic efficacy as monotherapy in some cancer models. Many different methods have been employed to improve the oncolytic potency of OVs. In this study, we used a directed evolution process, pooling different strains of VACV, including Copenhagen, Western Reserve and Wyeth strains and the attenuated modified vaccinia virus Ankara (MVA), to generate a new recombinant poxvirus with increased oncolytic properties. Through selective pressure, a chimeric VACV, deVV5, with increased cancer cell killing capacity and tumor selectivity in vitro was derived. The chimeric viral genome contains sequences of all parental strains. To further improve the tumor selectivity and anti-tumor activity of deVV5, we generated a thymidine kinase (TK)-deleted chimeric virus armed with the suicide gene FCU1. This TK-deleted virus, deVV5-fcu1 replicated efficiently in human tumor cells, and was notably attenuated in normal primary cells. These studies demonstrate the potential of directed evolution as an efficient way to generate recombinant poxviruses with increased oncolytic potency, and with high therapeutic index to improve cancer therapy.

Abstract LB-287: Pseudocowpox: A next generation viral vector for cancer immunotherapy

Engineered viral vectors are effective approaches to stimulate anti-tumor immunity, and change the tumor immune environment. Several viruses and strains have been developed to express tumor antigens and cytokines, and corresponding products are in advanced clinical trials. However, novel viral strains with improved immunogenic properties are sought. In this perspective, we screened a variety of poxviridae potentially usable in humans: Cowpox (CPX), Pseudocowpox (PCPV), Parapoxvirus Ovis (ORF), Myxoma virus (MYX), Swinepox (SWP), Yaba-like disease virus (YLDV), Raccoonpox (RCN), Cotia virus (CTV), and compared them to the well-established vaccine strain Modified Virus Ankara (MVA), and oncolytic Vaccinia Virus, strain Copenhagen (VV). Both in vitro with human primary immune cells, and in vivo with syngeneic mouse tumor models, PCPV proved to be a very promising vector for immunotherapy. Compared with MVA, PCPV induced a 1000-fold higher expression of IFN-alpha in human PBMCs, whereas SWPV and ORF displayed a lower 10 to 100-fold induction. Other viruses (i.e. VV, RCN, CTV, or MYX) did not raise the IFN-alpha level. PCPV was also shown to increase the level of GM-CSF, and to be safe for PBMCs, in contrast to ORF which displayed some cytotoxic effects. When tested for its capacity to trigger the expression of CD86, a co-stimulatory factor for T-cell activation, PCPV was shown to be superior than MVA in primary moDCs. Furthermore, PCPV treatment increased CD86 expression in human CD163+CD206+ “M2”-type macrophages derived from CD14+ monocytes, suggesting a shift to an antigen-presenting phenotype. In these cells, PCPV increased significantly the secretion of IL-18, IL-6 and IP-10, signing a conversion towards a less suppressive macrophage phenotype. Last, incubation of PCPV in a co-culture model overcame the immunosuppressive effect of MDSCs on human autologous CD8+ T. A GFP-encoding recombinant PCPV vector was generated, and we could demonstrate that PCPV was capable of infecting human primary immune cells, comparably to recombinant MVA vectors, except for activated T cells. A recombinant PCPV encoding for the HPV E7 protein was generated to assess the anti-tumor activity and immunogenicity in a syngeneic murine tumor model. Like MVA-E7, PCPV-E7 induced a strong cellular response (ELISPOT on splenocytes, and frequency of antigen-specific short-lived effector cells), but PCPV-E7 displayed a different cytokine/chemokine profile at the site of injection, with increased levels of pro-immune cytokines including IP-10, IFN-gamma, GM-CSF, IL-18, MIP-1 alpha, MIP-1 beta, IL-12 and IL-6. When injected intratumorally into fast growing MC-38 tumors, PCPV led to tumor control. Analysis of tumors infiltrates showed that PCPV treatment led to higher levels of neutrophils and decreased the frequency of MHCIIlo TAMs. Our data demonstrate that PCPV might display better properties than current viral vectors, in terms of local response and priming activity, of ability to induce effector T cells and to reshape the tumor infiltration profiles. It has the same capacity as other poxviruses to encode and deliver large genetic payload, which will be useful for designing advanced anti-tumor vaccines. Citation Format: Karola Rittner, Marine Ricordel, Caroline Tosch, Christine Thioudellet, Christelle Remy-Ziller, Marie-Christine Claudepierre, Chantal Hoffmann, Doris Schmitt, Benoit Sansas, Johann Foloppe, Philippe Erbs, Nathalie Silvestre, Kaidre Bendjama, Eric Quemeneur. Pseudocowpox: A next generation viral vector for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-287.

Abstract 4563: Local and abscopal effects in oncolytic virotherapy are boosted by immune checkpoint blockade, immunogenic chemotherapy, or IFNAR blockade

Athough the clinical efficacy of oncolytic viruses has been demonstrated for local treatment, the ability to induce immune-mediated regression of distant metastases is still poorly documented. We here report that an engineered oncolytic Vaccinia Virus, VVWR-TK-RR--Fcu1, is able to induce an immunogenic cell death and thus to generate a systemic immune response. Effect on tumor growth and survival is largely driven by CD8+ T-cells, and we could demonstrate that the immune cell infiltrate in the tumor could be reprogrammed towards a higher ratio of effector T-cells to regulatory CD4+ T-cells. The key role of the type 1-IFN pathway in oncolytic virotherapy was also highlighted, and we could show a strong abscopal response in Ifnar-/- tumors. In this model, the single administration of the virus directly into the tumors, on one flank, led to regression in the contralateral flank (i.e. opposite to the virus injection site). Moreover, we observed that these effects are further enhanced when the oncolytic treatment is combined with either immunogenic chemotherapy such as oxaliplatin, or with immune checkpoint blockers such as anti-PD-1 or anti-CTLA-4. Altogether, these data suggest that local oncolytic virotherapy in patients with tumors altered in IFNAR signaling could increase immune-mediated abscopal regression of distant metastases. Citation Format: Laetitia Fend, Takahiro Yamazaki, Xavier Preville, Eric Quemeneur, Oliver Kepp, Julien Adam, Aurelien Marabelle, Jonathan Pitt, Guido Kroemer, Laurence Zitvogel. Local and abscopal effects in oncolytic virotherapy are boosted by immune checkpoint blockade, immunogenic chemotherapy, or IFNAR blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4563. doi:10.1158/1538-7445.AM2017-4563

Abstract 2352: Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allow their intratumoral delivery and an improved tumor-growth inhibition

We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted in the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The 3 purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro. Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e. without tumor) confirming the virus tropism for tumoral cells and/or that tumoral microenvironment allows virus escape from immune surveillance. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mPD-1, than after IT administration of 10 μg of J43. Interestingly, in the MCA 205 tumor model, WR-mPD-1 (both mAb and scFv) induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that provided by an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs. New generation of oncolytic vaccinia virus that will express several transgenes simultaneously may also be designed with the goal of providing to the patients enhanced therapeutic/toxicity ratio. Citation Format: Jean-Baptiste Marchand, Patricia Kleinpeter, Laetitia Fend, Christine Thioudellet, Michel Geist, Nathalie Sfrontato, Veronique Koerper, Renee Brandely, Dominique Villeval, Karola Rittner, Nathalie Silvestre, Philippe Erbs, Laurence Zitvogel, Eric Quemeneur, Xavier Preville. Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allow their intratumoral delivery and an improved tumor-growth inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2352.